Substantial interest has been directed to the detection of changes in nucleic acid sequences, such as caused by mutation and methylation. For example, mutation in certain genes have been associated with a variety of disorders-ranging from blood disorders to cancers. Genetic testing is one way to find this information out. However, our ability to detect such mutations is limited by certain problems with a key component in these tests, namely the polymerase chain reaction (PCR).
A major problem with PCR is that polymerases invariably generate errors during amplification. Such polymerase misincorporations can be indistinguishable from genuine mutations, and lower the quality of DNA cloning and protein functional analysis by in vitro translation. Polymerase misincorporations set a limit for molecular mutation detection methods: the most selective technologies invariably rely on PCR, but PCR also poses a final selectivity limit, typically 1 mutant in 105-106 alleles, since all DNA polymerases generate errors during DNA synthesis which can be misinterpreted as mutations (false positives). Thus, high selectivity mutation detection technologies often fall short of the enormous selectivity needed to address issues like the generation of spontaneous mutations in somatic tissues1,2, the early detection of genomic instability3, the mutation screening of single cells4 or the reliable detection of minimal residual disease5, 6. Both unknown and known mutation detection methods are affected by PCR errors and the most selective methods are affected most.
For example, the principal limitation for mutation scanning via constant denaturant capillary electrophoresis (CDCE) is the fidelity of the polymerase used7, 8. High selectivity mutation scanning via DGGE and dHPLC is ultimately hindered by polymerase error rate7, 9, 10. Some of the highest sensitivity assays for RFLP-based known mutation detection, including PCR/RE/LCR11, MutEx-ACB-PCR12, Radioactivity-based PCR-RFLP13, RSM14, 15, APRIL-ATM16, and others reviewed in Parsons et al.17, utilize PCR in at least one stage prior to RFLP-selection, and are therefore also limited by PCR errors18.
Accordingly, it would be desirable if one had a means of amplifying DNA free of polymerase-induced misincorporations, to detect mutations without being limited by polymerase-induced errors. This could significantly impact mutation detection, disease diagnosis, and cancer diagnosis.